Frequently, a limiting factor for the therapeutic utilization of molecules whose target is within the cell is their unsatisfactory cellular uptake and unfavorable intracellular distribution. Typical examples are macromolecules such as nucleic acids, which bind in sequence-specific manner to cellular DNA or RNA, thus inhibiting gene expression. Antisense oligonucleotides are short single-stranded nucleic acids, which bind via Watson-Crick base pairs to complementary mRNA, inhibiting its translation into the corresponding protein. Triplex-forming oligonucleotides bind via so-called “Hoogsteen base pairing” to the deep groove of the DNA double helix forming a triple helix, thus inhibiting the transcription of the genes in a sequence-specific manner. Other intracellularly acting oligonucleotides are, for example, the so-called “decoy” oligonucleotides which mimic the binding regions for transcription factors. By treatment with decoy oligonucleotides, certain transcription factors can be intercepted in a sequence-specific manner, thus inhibiting activation of the transcription. A further group of intracellularly acting oligonucleotides, the chimera plasts, is used for targeted gene correction (Cole-Strauss et al., Science 273:1386-1389 (1996)). For this gene correction, too, the uptake of the chimera plast oligonucleotide into the cell is essential. Examples of further intracellularly acting nucleic acids are those which interact with cellular enzymes, in particular with telomerases (Norton et al. Nat. Biotechn. 14:615 (1996)). A further class of nucleic acids, preferably double-stranded DNA, can code for certain proteins, which are expressed intracellularly in the sense of gene therapy.
For example, the uptake of an oligonucleotide in vitro into a cell, for example by simple addition of the oligonucleotide to the cell culture medium, is a relatively inefficient process, because only a small fraction of the added oligonucleotide is actually taken up into the cell. The uptake process takes many hours, and in most cases, a plateau phase is reached only after 8 to 16 hours. It is assumed that the oligonucleotides are taken up in an endocytosis-like process. However, a general problem with uptake via endocytosis is that a large proportion of the oligonucleotides present are not free in the cytoplasm, but enclosed in certain cell structures, i.e., the lysosomes and endosomes. In the case of fluorescently labeled oligonucleotides, this localized distribution can indeed be observed by fluorescence microscopy. Owing to this vesicular localization, the concentration of free oligonucleotide, which is actually available for hybridization to the mRNA, is considerably reduced. Moreover, depending on the cell type and the conditions present, only a certain fraction of cells take up the oligonucleotide in the first place. Therefore, for the efficient use of antisense oligonucleotides, mixtures with penetration enhancers, such as, for example, cationic lipids (Bennett et al., Mol. Pharmacol. 41:1023 (1992)) are generally employed.
It was an object of the present invention to improve cellular uptake of molecules, in particular of macromolecules, such as, for example, oligonucleotides.
Examination of cellular uptake of oligonucleotides is generally carried out using either radioactively labeled or fluorescently labeled oligonucleotides. Fluorescence labeling of an oligonucleotide is carried out, for example, by reacting the amino function of an oligonucleotide with fluorescein isothiocyanate (FITC). The fluorescein can be introduced, for example, into the 3′ end of an oligonucleotide via a commercially available fluorescein-derivatized solid-phase support, or into the 5′ end via a commercially available fluorescein phosphitylating reagent. In all cases, the oligonucleotide-bound fluorescein is, owing to the carboxylic acid function, present as a negatively charged structural element, which is strongly fluorescent.

In contrast to fluorescein, fluorescein diacetate (FDA) is a neutral vital dye, which is transformed into the fluorescent fluorescein only after removal of the two ester groups and opening of the lactone ring, but which is not fluorescent in the form of the lactone.
It is known that FDA (hereinbelow also referred to as “F3”), as a neutral, non-fluorescent molecule, is taken up by living cells via passive diffusion and is cleaved intracellularly by esterases to give the fluorescent fluorescein (Breeuwer et al., Appl. Environ. Microbiol. 61:1614 (1995); Maeda et al., Cell Struct. Funct. 7:177 (1982)). Hitherto, the only FDA derivatives described have been those containing an amine-reactive group, such as, for example, isothiocyanate; these FDA derivatives are used for staining intracellular proteins or cell components. Neither conjugates of FDA with other molecules nor FDA-labeled oligonucleotides (conjugates of FDA and oligonucleotide) have been described-previously.
In the cytoplasm, FDA is cleaved by esterases. Accordingly, it is possible to determine, by FDA labeling of an oligonucleotide, the proportion of “free” oligonucleotide, i.e., how much oligonucleotide is present in the cytoplasm and available for hybridization—in relation to the proportion of oligonucleotide present in vesicles (“captured” oligonucleotide)—and accordingly not available for hybridization. Owing to the high total number of negative charges in an oligonucleotide and the fact that FDA-labeled and fluorescein-labeled oligonucleotides (in the case where the oligonucleotide is identical) differ by only one net charge, one would expect that FDA-labeled and fluorescein-labeled oligonucleotides would exhibit very similar cellular uptake and distribution.
However, surprisingly, it has been found that FDA-labeled and fluorescein-labeled oligonucleotides differ considerably in their uptake into cells, i.e., in duration and efficiency of the uptake of the oligonucleotides as well as in cellular localization of the oligonucleotides that have been taken up. Cells take up FDA-labeled oligonucleotide much more rapidly than the corresponding fluorescein labeled oligonucleotide. FDA-labeled oligonucleotides can, after simple incubation, for example with human cells, be detected intracellularly after only five minutes, whereas, the uptake of radioactively labeled and fluorescein-labeled oligonucleotides requires several hours. It is also surprising that the FDA-labeled oligonucleotides are taken up into virtually any cells (>90% of cells). Whereas, the rate of uptake in the methods hitherto described for transferring oligonucleotides or polynucleotides into cells is generally considerably lower; in the latter case, frequently only about 30 to 60% of the cells are loaded with oligonucleotides. Another advantage is the intracellular distribution of the FDA-labeled oligonucleotides, which is much more uniform. This more uniform distribution indicates that the oligonucleotides are not—as described above—mainly enclosed in vesicles (for example, endosomes, lysosomes), but distributed in the entire cell—i.e., in the cytosol and the nucleus. This is an indication that a large fraction of “free” oligonucleotide is present. Only these “free” oligonucleotides are available for binding to the target (target molecule, target nucleic acid) or as active compound. Another advantage is the fact that no damage to the cells is observed when FDA-labeled oligonucleotides are used; in contrast, the use of lipocationic penetration enhancers frequently results in damage of the cell membrane. As a consequence of these unexpected properties, the FDA-labeled oligonucleotides have, compared to the methods hitherto described for introducing oligonucleotides or polynucleotides into cells, the decisive advantage that they can be introduced into the cells more effectively, where they are also more readily available. Owing to this, the FDA-labeled oligonucleotides have a considerably improved biological activity. Because of the improved biological activity, less oligonucleotide has to be used. Owing to this and the fact that a FDA-labeled oligonucleotide is taken up more effectively—both with respect to the amount and to time—into a cell, (toxic) side effects are reduced.
Surprisingly, it has been found that these advantageous properties are not limited to FDA-labeled oligonucleotides. Virtually any molecule can be introduced effectively into a cell or transported across a biological membrane with the aid of FDA-labeling—i.e., by coupling a molecule to be transported to FDA, or conjugating it (“FDA conjugate”). Furthermore, it has been found that this principle is not limited to FDA conjugates, but also applies to all aryl ester conjugates of a certain chemical structure. Thus, the present invention is a novel principle for transporting molecules across biological membranes. Since these compounds have hitherto, except for one exception, not been described in the prior art, the corresponding conjugates—a molecule to be transported coupled to or conjugated with an aryl ester of a certain chemical structure—likewise form part of the subject matter of the present invention. These conjugates cannot be prepared by known processes. The present invention, therefore, also provides a process for preparing the conjugates.
Bioreversible O-acylaryl conjugates, which have been proposed as prodrugs of oligonucleotides (lyer et al., Bioorganic & Med. Chem. Lett. 7: 871-876 (1997)), are known. The chemical structure of these compounds is—in the case that the aryl radical is an aromatic 6-membered ring—similar to that of the conjugates according to the invention. However, in the bioreversible O-acylaryl conjugates, the hydrolysis of the ester results in a destabilization of the bond between the aryl radical and the phosphotriester of the oligonucleotide, so that the bioreversible O-acylaryl conjugate is cleaved into its components, i.e., the free oligonucleotide and the O-acylaryl radical. This prodrug concept serves to mask the negative charge of the internucleotide phosphate bridge and thus, to facilitate uptake of the oligonucleotide into the cell. However, in contrast to the conjugates according to the invention, no accelerated uptake of the oligonucleotides into the cells and likewise no changed intracellular distribution of the oligonucleotides have been found for these prodrugs. Furthermore, an uptake of the oligonucleotides into other organisms has not been reported. In contrast, in the conjugates according to the invention, the covalent bond between the aryl radical and the oligonucleotide is preserved during uptake into the cell. The preservation of the covalent bond between aryl radical and oligonucleotide can easily be determined by fluorescence microscopy, if the aromatic unit is only fluorescent after cleavage of the ester, such as, for example, in the case of FDA.